Fluorescent dyes are widely used for biological, biochemical or chemical applications in which a highly sensitive detection reagent is desirable. Molecules labeled with sensitive dye reagents enable the researcher to determine the presence, quantity or location of such molecules by monitoring their fluorescence. The quenching and energy transfer properties of fluorescent dyes also render the dyes useful for a variety of methods permitting investigators to monitor the in vivo and in vitro interactions between labeled molecules (e.g., protein:protein, protein:nucleic acid, nucleic acid:nucleic acid, and protein: or nucleic acid: interactions with drugs, drug candidates or other chemical entities). Fluorescent dyes are also useful for non-biological applications, such as photographic media.
In comparison to radiolabels frequently used for similar purposes, fluorescent dyes have the advantages of being safe to handle and dispose of and relatively stable. In addition, fluorescent dyes are detectable in real time, and differing fluorescence excitation and emission spectra can be exploited to permit differential detection of two, three or more different labeled species in a given mixture.
There is a wide variety of fluorescent dyes known and used in various fields. One class, the cyanine dyes, is very frequently used in biological and biochemical applications. Cyanine dyes are generally characterized by the presence of a pair of nitrogen-containing heterocycles (xe2x80x9cterminal heterocyclesxe2x80x9d) connected by a polymethine bridge over which bond resonance occurs. Most cyanine dyes exhibit high visible absorbance and reasonable resistance to photodegradation. The general structure of cyanine dyes is given by the following formula: 
In the above formula, X and Y are typically heteroatoms (e.g., O, S, N) or disubstituted carbon atoms (e.g., Cxe2x80x94(CH3)2). Those dyes wherein n=0 are typically referred to as xe2x80x9ccyaninexe2x80x9d dyes. Where n=1 the dyes are termed xe2x80x9ccarbocyaninexe2x80x9d dyes, while where n=2 the dyes are xe2x80x9cdicarbocyaninexe2x80x9d dyes and if n=3 the dyes are xe2x80x9ctricarbocyaninexe2x80x9d dyes, and so on. This class of dyes is generically referred to as xe2x80x9ccyaninexe2x80x9d dyes regardless of the specific number of methine groups between the ring systems.
The substituents R1 and R2 are typically saturated or unsaturated alkyl groups that are optionally further substituted by a wide variety of other functional groups. Other positions on the terminal heterocycles may be substituted with various organic functional groups, as well as additional fused or unfused rings that may themselves be additionally substituted.
There is a need in the art for additional fluorescent dyes. In particular, there is a need for dyes with fluorescence characteristics that permit distinct detection in multiple labeling assays, both in vitro and in vivo. Dye characteristics that can be altered to advantage include, for example, fluorescence excitation and emission spectra, fluorescence efficiency and quantum yield, fluorescence intensity, and characteristics such as solubility, chemical stability and compatibility with given assay conditions (e.g., in vitro, in vivo, aqueous, non-aqueous, etc.).
Fluorescence characteristics of the cyanine dyes can be altered, for example, by changing the aromatic nature of, or substituents on, the terminal heterocycles, or by changing the number of methine groups between the aromatic moieties. Generally, the longer the polymethine bridge, the higher the wavelengths of excitation and emission (i.e., longer polymethine bridges tend to shift excitation and emission spectra to the red, a so-called xe2x80x9cred shiftxe2x80x9d). However, in general, the stability of the dye and the fluorescence efficiency decreases with increasing polymethine bridge length. It is desirable to alter fluorescence characteristics without dramatically increasing the size of the polymethine bridge.
The invention relates to novel fluorescent cyanine dyes and to molecules labeled with them. The dyes according to the invention are water soluble and can be used in any application normally requiring water soluble fluorescent dyes. The invention encompasses compositions comprising the novel fluorescent cyanine dyes disclosed herein, as well as nucleosides, nucleotides, polynucleotides, or other molecules or biomolecules labeled with such dyes.
In one aspect, fluorescent cyanine dyes according to the invention have a hetero cyclic structure integrated into the characteristic polymethine bridge between the terminal heterocycles that are characteristic of cyanine dyes. Dyes according to this aspect of the invention have longer emission wavelengths than dyes having similar terminal heterocycles but lacking the heterocycle integrated into the polymethine bridge.
In other aspects, the fluorescent cyanine dyes according to the invention have novel arrangements of functional groups for linkage to molecules of interest, novel combinations or arrangements of terminal heterocycles and/or structures providing structural rigidity. Each of the dyes according to these aspect of the invention has different fluorescence characteristics, making them useful for multiparameter assays and/or assays based on energy transfer.
The invention encompasses a fluorescent cyanine dye having the formula:
T1(xe2x80x94CHxe2x95x90)n1A(xe2x80x94CHxe2x95x90)n2T2
wherein: nxe2x89xa71 and n1 is the same as or different from n2; A comprises the formula: 
wherein: X1 and Y1 are selected from the group consisting of C(CH3)2, CHxe2x95x90CH, O, N, S, Se and Te and either X1 or Y1 is N; X2 and Y2 are selected from the group consisting of C(CH3)2, CHxe2x95x90CH, O, N, S, Se and Te and either X2 or Y2 is N; or A comprises the formula: 
wherein: Z1 and Y1 are selected from the group consisting of C(CH3)2, CHxe2x95x90CH, O, N, S, Se and Te and either Z1 or Y1 is N; Z2 and Y2 are selected from the group consisting of C(CH3)2, CHxe2x95x90CH, O, N, S, Se and Te and either Z2 or Y2 is N; and wherein a and b are 0 or 1, and a+b=1; and where X, Y or Z is N, R2 and R3 are substituents on N and are the same or different and are selected from the group consisting of H, methyl, ethyl, C(CH3)2 and (CH2)qV, wherein q is an integer from 1 to 25 and V is a reactive group or H; and wherein T1 and T2 are the same or different and have the formula: 
wherein: Q is selected from the group consisting of O, S, CH2, (CHxe2x95x90CH) and C(CH3)2; R1 and R4 are the same or different and are selected from the group consisting of H, methyl, ethyl and (CH2)qV, wherein q is an integer from 1 to 25 and V is a reactive group or H; each of W1-8 is the same or different and may be H or a hydrophilic moiety; at least one occurrence of W is a hydrophilic moiety; and wherein at least one of R1-R4 has a reactive group.
In one embodiment, one or both of Y1 and Y2 are N. It is preferred that in the dye of this embodiment, one or both of X1 and X2 are S. It is also preferred in this embodiment that one or both of X1 and X2 are O. It is also preferred in this embodiment that one or both of X1 and X2 are CH2. It is also preferred in this embodiment that one or both of X1 and X2 are (CHxe2x95x90CH). It is also preferred in this embodiment that one or both of Y1 and Y2 are S.
In another embodiment, Z1 and Y2 are S.
In another embodiment, Y1 and Z2 are S.
In another embodiment, Q is CH2.
In another embodiment, Q is C(CH3)2.
The invention further encompasses a composition comprising a dye as described above.
The invention further encompasses a fluorescent cyanine dye having the formula: 
wherein: nxe2x89xa71; Q is selected from the group consisting of O, S, CH2, (CHxe2x95x90CH) and C(CH3)2; R1-R4 are the same or different and are selected from the group consisting of H, methyl, ethyl and (CH2)qV, wherein q is an integer from 1 to 25 and V is a reactive group or H, and at least one of R1-R4 has a reactive group; each of W1-8 is the same or different and may be H or a hydrophilic moiety; and at least one occurrence of W is a hydrophilic moiety. The invention also encompasses a composition comprising such a dye.
The invention further encompasses a fluorescent cyanine dye having the formula: 
wherein R1-R4 are the same or different and are selected from the group consisting of H, methyl, ethyl and (CH2)qV, wherein q is an integer from 1 to 25 and V is a reactive group or H, and at least one of R1-R4 has a reactive group. The invention further encompasses a composition comprising such a dye.
The invention further encompasses a fluorescent cyanine dye having the formula: 
wherein: nxe2x89xa71; Q is selected from the group consisting of O, S, CH2, (CHxe2x95x90CH) and C(CH3)2; R1-R4 are the same or different and are selected from the group consisting of H, methyl, ethyl and (CH2)qV, wherein q is an integer from 1 to 25 and V is a reactive group or H, and at least one of R1-R4 has a reactive group; each of W1-8 is the same or different and may be H or a hydrophilic moiety; and at least one occurrence of W is a hydrophilic moiety. The invention further encompasses a composition comprising such a dye.
The invention further encompasses a fluorescent cyanine dye having the formula: 
wherein R1-R4 are the same or different and are selected from the group consisting of H, methyl, ethyl and (CH2 )qV, wherein q is an integer from 1 to 25 and V is a reactive group or H, and at least one of R1-R4 has a reactive group. The invention further encompasses a composition comprising such a dye.
The invention further encompasses a fluorescent cyanine dye having the formula: 
wherein: nxe2x89xa71; Q is selected from the group consisting of O, S, CH2, (CHxe2x95x90CH) and C(CH3)2; R1-R4 are the same or different and are selected from the group consisting of H, methyl, ethyl and (CH2)qV, wherein q is an integer from 1 to 25 and V is a reactive group or H, and at least one of R1-R4 has a reactive group; each of W1-8 is the same or different and maybe H or a hydrophilic moiety; and at least one occurrence of W is a hydrophilic moiety. The invention further encompasses a composition comprising such a dye.
The invention further encompasses a fluorescent cyanine dye having the formula: 
wherein R1-R4 are the same or different and are selected from the group consisting of H, methyl, ethyl and (CH2)qV, wherein q is an integer from 1 to 25 and V is a reactive group or H, and at least one of R1-R4 has a reactive group. The invention further encompasses a composition comprising such a dye.
The invention further encompasses a fluorescent cyanine dye having the formula: 
wherein: nxe2x89xa71; Q is selected from the group consisting of O, S, CH2, (CHxe2x95x90CH) and C(CH3)2; R1-R4 are the same or different and are selected from the group consisting of H, methyl, ethyl and (CH2)qV, wherein q is an integer from 1 to 25 and V is a reactive group or H, and at least one of R1-R4 has a reactive group; each of W1-8 is the same or different and may be H or a hydrophilic moiety; and at least one occurrence of W is a hydrophilic moiety. The invention further encompasses a composition comprising such a dye.
The invention further encompasses a fluorescent cyanine dye having the formula: 
wherein R1-R4 are the same or different and are selected from the group consisting of H, methyl, ethyl and (CH2)qV, wherein q is an integer from 1 to 25 and V is a reactive group or H, and at least one of R1-R4 has a reactive group. The invention further encompasses a composition comprising such a dye.
The invention further encompasses a fluorescent cyanine dye having the formula: 
wherein: nxe2x89xa71; Q is selected from the group consisting of O, S, CH2, (CHxe2x95x90CH) and C(CH3)2; R1-R4 are the same or different and are selected from the group consisting of H, methyl, ethyl and (CH2)qV, wherein q is an integer from 1 to 25 and V is a reactive group or H, and at least one of R1-R4 has a reactive group; each of W1-8 is the same or different and may be H or a hydrophilic moiety; and at least one occurrence of W is a hydrophilic moiety. The invention further encompasses a composition comprising such a dye.
The invention further encompasses a fluorescent cyanine dye having the formula: 
Wherein R1-R4 are the same or different and are selected from the group consisting of H, methyl, ethyl, C(CH3)2 and (CH2)qV, wherein q is an integer from 1 to 25 and V is a reactive group or H, and at least one of R1-R4 has a reactive group. The invention further encompasses a composition comprising such a dye.
The invention further encompasses a fluorescent cyanine dye having the formula: 
Wherein: nxe2x89xa71; Q is selected from the group consisting of O, S, CH2, (CHxe2x95x90CH) and C(CH3)2; R1-R4 are the same or different and are selected from the group consisting of H, methyl, ethyl and (CH2)qV, wherein q is an integer from 1 to 25 and V is a reactive group or H, and at least one of R1-R4 has a reactive group; each of W1-8 is the same or different and may be H or a hydrophilic moiety; and at least one occurrence of W is a hydrophilic moiety. The invention further encompasses a composition comprising such a dye.
The invention further encompasses a fluorescent cyanine dye having the formula: 
wherein R1-R4are the same or different and are selected from the group consisting of H, methyl, ethyl and (CH2)qV, wherein q is an integer from 1 to 25 and V is a reactive group or H, and at least one of R1-R4 has a reactive group. The invention further encompasses a composition comprising such a dye.
The invention further encompasses a fluorescent cyanine dye having the formula: 
wherein: nxe2x89xa71; Q is selected from the group consisting of O, S, CH2, (CHxe2x95x90CH) and C(CH3)2; R1-R4 are the same or different and are selected from the group consisting of H, methyl, ethyl and (CH2)qV, wherein q is an integer from 1 to 25 and V is a reactive group or H, and at least one of R1-R4 has a reactive group; each of W1-8 is the same or different and may be H or a hydrophilic moiety; and at least one occurrence of W is a hydrophilic moiety. The invention further encompasses a composition comprising such a dye.
The invention further encompasses a fluorescent cyanine dye having the formula: 
wherein R1-R4 are the same or different and are selected from the group consisting of H, methyl, ethyl and (CH2)qV, wherein q is an integer from 1 to 25 and V is a reactive group or H, and at least one of R1-R4 has a reactive group. The invention further encompasses a composition comprising such a dye.
The invention further encompasses a fluorescent cyanine dye as disclosed herein, wherein the dye further comprises a succinimide ester linked to a heterocyclic nitrogen.
The invention further encompasses a nucleoside or nucleotide labeled with a dye as disclosed herein.
The invention further encompasses a polynucleotide labeled with a dye as disclosed herein.
The invention further encompasses a polypeptide labeled with a flourescent cyanine dye as disclosed herein.
The invention further encompasses a method of labeling a nucleotide or nucleoside, the method comprising contacting a fluorescent cyanine dye of the invention with the nucleotide or nucleoside. In one embodiment, the dye comprises a reactive group and the nucleotide or nucleoside comprises a functional group complementary to the reactive group, wherein the contacting results in the covalent attachment of the dye to the nucleotide or nucleoside. In another embodiment, wherein the nucleotide or nucleoside comprises a reactive group and the dye comprises a functional group complementary to the reactive group, wherein the contacting results in the covalent attachment of the dye to the nucleotide or nucleoside.
The invention further encompasses a method of labeling a nucleic acid, the method comprising contacting a fluorescent cyanine dye of the invention with the nucleic acid. In one embodiment, the nucleic acid comprises a reactive group and the dye comprises a functional group complementary to the reactive group, wherein the contacting results in the covalent attachment of the dye to the nucleic acid. In another embodiment, the dye comprises a reactive group and the nucleic acid comprises a functional group complementary to the reactive group. In another embodiment, the nucleic acid comprises an allyl-amine-modified nucleotide, and the dye comprises an NHS group.
The invention further encompasses a method of labeling a polypeptide, the method comprising contacting a fluorescent cyanine dye of the invention with the polypeptide. In one embodiment, the method comprises contacting a polypeptide comprising a reactive group with a fluorescent cyanine dye of the invention, wherein the dye comprises a functional group complementary to the reactive group, and wherein the contacting results in the covalent attachment of the dye to the polypeptide.
The invention further encompasses a method of labeling a nucleic acid, the method comprising contacting the nucleic acid with a cis-platinum complex comprising a fluorescent cyanine dye of the invention.
The invention further encompasses a method of determining a nucleic acid sequence, the method comprising performing a nucleic acid sequencing reaction in the presence of a nucleotide labeled with a fluorescent cyanine dye of the invention. In one embodiment, the sequencing reaction is performed in the presence of a second nucleotide comprising a fluorescent dye that is spectrally distinct from the dye on the first nucleotide.
The invention further encompasses a method of determining a nucleic acid sequence, the method comprising determining a nucleic acid sequence on a nucleic acid comprising a fluorescent cyanine dye of the invention.
The invention further encompasses a method of detecting a polynucleotide, the method comprising detecting a polynucleotide comprising a nucleotide labeled with a fluorescent cyanine dye of the invention.
The invention further encompasses a method of detecting a polynucleotide, the method comprising detecting a polynucleotide comprising a fluorescent cyanine dye of the invention. In one embodiment, the method comprises hybridizing a nucleic acid probe comprising sequence complementary to the polynucleotide, wherein the nucleic acid probe comprises a fluorescent cyanine dye of the invention. In one embodiment, the detecting is performed on a nucleic acid microarray.
The invention further encompasses a method of detecting a polypeptide, the method comprising detecting a polypeptide comprising a fluorescent cyanine dye of the invention.
As used herein, the term xe2x80x9cfluorescentxe2x80x9d refers to the property of a molecule whereby, upon irradiation with light of a given wavelength or wavelengths, the molecule becomes excited and emits light of a longer wavelength or wavelengths. The term xe2x80x9cfluorophorexe2x80x9d as used herein refers to a fluorescent molecule. There are a number of parameters which together describe the fluorescence characteristics of a fluorophore. These include, for example, the maximum wavelengths of excitation and emission, the breadth of the peaks for excitation and emission, the difference between the excitation and emission maxima (the xe2x80x9cStokes shiftxe2x80x9d), fluorescence intensity, quantum yield, and extinction coefficient. For biological or biochemical applications, longer Stokes shifts are generally preferred to shorter ones.
Fluorescence intensity is determined as the product of the extinction coefficient and the fluorescence quantum yield. The fluorescence quantum yield is a measure of the relative efficiency or extent to which light energy absorbed is re-emitted as fluorescence. It is defined as the ratio of the number of fluorescence photons emitted, F to the number of photons absorbed, A, and molecules with larger quantum yields exhibit greater fluorescence intensity.
The molar extinction coefficient is a measure of a fluorophore""s ability to absorb light. Commonly used fluorophores tend to have molar extinction coefficients (at their absorption maximum) between 5,000 and 200,000 cmxe2x88x921Mxe2x88x921 (Haugland, R. P. (1996) Molecular Probes Handbook for Fluorescent Probes and Research Chemicals, 6th Edition). Because fluorescence intensity is the product of quantum yield and the extinction coefficient, higher extinction coefficients also correlate with greater fluorescence intensity.
As used herein, the term xe2x80x9cwater solublexe2x80x9d refers to a composition which dissolves in water. In order to be termed xe2x80x9cwater solublexe2x80x9d according to the invention, a fluorescent dye will form at least a 5 mM solution in water at room temperature (about 21xc2x0 C.) and neutral pH. A water soluble fluorescent dye will preferably dissolve in water at neutral pH and room temperature to generate a solution that is at least 10 mM, 20 mM, 50 mM, 100 mM, 250 mM, 500 mM or more, up to and exceeding 1 M or more. The water solubility of a dye according to the invention may be increased by the addition of xe2x80x9chydrophilic moietiesxe2x80x9d at one or more sites on the molecule. xe2x80x9cHydrophilic moietiesxe2x80x9d are defined herein as polar or electrically charged groups that increase the water solubility of the dye molecule relative to the same dye molecule without the moiety. Examples of hydrophilic moieties include sulfonate, hydroxy, sulfate, sulfonate, carboxylate, phosphate, phosphonate, substituted amino or quaternary amino groups, or any of these groups attached to a lower alkyl group (lower alkyl groups have 10 or fewer carbon atoms).
In some embodiments, sulfonate groups are present on dyes according to the invention, increasing their water solubility. In other embodiments, however, the dyes lack sulfonate groups yet contain ionizable carboxylate (COOxe2x88x92) moieties which confer water solubility on the free dye while also providing a convenient site for attachment to a biomolecule. The carboxylate group loses its charge upon attachment to a (water soluble) biomolecule (becoming an amide or peptide bond), however the final product retains water solubility as defined herein.
As used herein, the term xe2x80x9cterminal heterocyclexe2x80x9d refers to a nitrogen-substituted heterocycle located on either end of a polymethine bridge in a cyanine dye. In a dye according to the invention, at least one, and preferably both of the terminal heterocycles will comprise a quaternary nitrogen at the site of attachment to the polymethine bridge.
As used herein, the term xe2x80x9creactive groupxe2x80x9d refers to a chemical group which will react with a complementary functional group on a molecule of interest to form a covalent bond between the fluorescent dye to the molecule of interest. Reactive groups useful according to the invention include halomethyl (xe2x80x94CH2xe2x80x94X), haloacetamide (xe2x80x94NHxe2x80x94(Cxe2x95x90O)xe2x80x94CH2xe2x80x94X), halomethylbenzamide (xe2x80x94NHxe2x80x94(Cxe2x95x90O)xe2x80x94C6H4xe2x80x94CH2xe2x80x94X) where X is Cl, Br or I. Reactive groups also include, but are not limited to an amine, a carboxyl, a thiol, maleimide, an azido, a (3,5-dichloro-2,4,6-triazin-1-yl) amino, an isocyanato, an isothiocyanato, an acyl halide, a succinimidyl ester, a sulfosuccinimidyl ester, a pentafluorophenyl, a 4-sulfotetrafluorophenyl, or a hydroxybenzotriazole group, among others.
As used herein, the term xe2x80x9cpolymethine bridgexe2x80x9d refers to a chain of covalently bonded carbon atoms having alternating single and double bonds between them (e.g., xe2x80x94CHxe2x95x90CHxe2x80x94CHxe2x95x90 . . . ; the methine group is xe2x95x90CHxe2x80x94), such that bond resonance between the single and double bonded state occurs over the length of the chain. A polymethine bridge joins nitrogens (at least one of which is, and preferably both of which are quaternary nitrogen(s)) comprised by two terminal heterocycles in a fluorescent dye according to the invention and will comprise at least two methine groups or at least four bonds which resonate between single and double bonded states. The polymethine bridge of a dye according to the invention can have substituted or unsubstituted cyclic structures comprising one or more rings integrated into it, as long as the bond resonance can still occur over the length of the whole bridge structure. The polymethine bridge structures of the dyes of the invention can confer structural stability on the dye, but their primary function is to allow electron resonance between the terminal heterocycle nitrogens joined by the polymethine bridge. This electron resonance permits absorbed excitation energy to be transmitted from one end of the fluorophore to the other during the process leading to fluorescent emission.
As used herein, the term xe2x80x9cnucleotidexe2x80x9d encompasses naturally occurring nucleotides, such as dATP, dCTP, dGTP, TTP, UTP, rATP, rCTP, rGTP, as well as synthetic or modified nucleotides such as ITP, and ddATP, ddCTP, ddGTP and ddTTP.
As used herein, the term xe2x80x9cpolynucleotidexe2x80x9d refers to a double or single stranded chain of two or more phosphodiester-linked nucleotides. The term encompasses DNA, RNA, peptide nucleic acids (PNA) and heteroduplexes of these, as well as synthetic combination polynucleotides comprising one or more of DNA, RNA or a PNA chemically linked in one molecule.
As used herein, the term polypeptide refers to a molecule consisting of two or more amino acids joined by peptide bonds.